Allergen-microarray assay

ABSTRACT

A method is provided for the detection of an immunoglobulin which binds to an allergen in a sample, whereby one or more allergens are immobilized on a microarray chip after which the sample is incubated with the immobilized allergens so that immunoglobulins which are specific for the allergens bind to the specific allergen after which the immunoglobulins which are bound to the specific immobilized allergens are detected, as well as a method for in vitro diagnosis of allergies in a patient.

The present invention relates to a method for the detection of animmunoglobulin which binds to an allergen in a sample as well as amethod for in vitro diagnosis of allergies in a patient.

An allergy is a reaction produced by the body's immune system to asubstance that would normally be thought of as harmless. It is thisresponse that causes the symptoms that are classed as allergicreactions. Allergy is therefore not a failure of the immune system, butits over activity. The response of an allergic person to an allergen canproduce a wide range of symptoms. Some people suffer symptoms such asasthma, eczema, rashes, itchy eyes, sinusitis, blocked or runny nose andhay fever, however, more serious symptoms can occur. With allergies suchas those to venoms, nuts and shellfish, for example, a potentially lifethreatening condition called anaphylactic shock can occur. This happenswhen the body produces a reaction so severe that the throat swells,blood pressure drops and the person has difficulty in breathing. In somecases this type of reaction can be fatal.

The incidence of allergies is increasing in the developed countries(i.e. in Europe, Northern America and Japan). Estimates range from20-50% of the population being affected. It is now known that allergiesare the result of an unbalance in the T-cell compartment of theimmune-system. More precisely, allergies are accompanied by an increasedactivity of so-called T-helper 2 (Th2) cells relative to T-helper 1(Th1) cells, giving rise to increased IgE production.

IgE (immunoglobulin E) is at least one of the substances that causeallergic reactions. IgE specific for an allergen is not normallydetected in the blood and is only produced when a person becomessensitised to a substance. Substances that cause an allergy (called anallergen) produce a specific IgE that is unique to and will only reactwith it. This reaction between IgE and the allergen is like a lock andkey. IgE, when combined with the allergen, causes cells to releasechemicals (e.g. histamine), which cause the symptoms of allergy. Aperson may have specific IgE to more than one substance and maytherefore be allergic to more than one substance. Results for IgEtesting are expressed as a grade that indicates how much IgE specific tothe substance tested for, is present in the blood. The higher the gradethe more likely the patient is to be allergic. Over the past decadesallergic diseases have been diagnosed using only scarcely purifiedextracts derived from the major allergen sources. Evidently, these crudemixtures are complex and variable in composition. As a consequence, thediagnostic potential of these extracts may vary and lead to theproduction of false-negative results. The latter has been ascribedmainly to the instability of allergens in extracts. Another drawback ofusing extracts in the diagnosis of allergies is the poor standardisationof commercially available products with respect to their allergeniccomposition. Units that express the potencies and methods of calculationare different for each company in the market. As a consequence,diagnostic products from different companies can hardly be compared.

The most important disease-related components, the major allergens, havebeen identified over the past decades, but their quantification is notused as the basis for standardisation of allergen extracts. Monoclonalantibodies against most major allergens are now available.

Allergy testing according to known methods is not a straightforwardprocess. These methods can be useful procedures provided that a historyis taken to identify which tests would be most appropriate. However,there are only very few recognised medical tests that can identifyallergies and as a general rule these have been restricted to clinicallaboratories or specialist centres. Whilst the diagnosis of atopy orallergy by a qualified practitioner or specialist in allergy can berelatively easily made further tests are often required to confirm this.

There are various types of allergy testing:

1. Skin Prick Testing

Suspected allergens are injected just under the surface of the skin andthe reaction is observed by a qualified nurse or doctor. As the reactiondevelops there is a zone of redness, the stronger the reaction thegreater the zone. Skin testing is quite sensitive although not allallergies can be identified by this method.

2. Patch Testing

Patch testing may be useful in cases of contact dermatitis. Testsubstances are usually applied to the skin covered by a patch and leftin place for 48 hours. A positive reaction produces a small area ofeczema. Again the reaction is observed by a qualified nurse or doctor.

3. Alternative Allergy Testing

There are many alternative types of allergy testing and unfortunatelyfew are accurate or recognised by the medical profession. Thesealternative tests are often misleading as well as costly.

4. Vega Test

The Vega test is an electrical test described as bioenergetic regulatorytechnique. The machine measures conductivity with a Wheatstone circuit.Electrodes are connected to acupuncture points or held in the patient'shand. Different solutions are then placed in a metallic tray. Themachine is then calibrated by placing a glass vial containing a toxicsubstance into the tray. The vial causes a reduction in electricalconductivity. Other substances are then placed into the tray and if theygive a similar reading this is reported as an allergic or “sensitive”reaction. This technique is widely used in health food stores however,its value is unproved and there are no valid trials that willsubstantiate claims.

5. Leucocytotoxic Test

The Leucocytotoxic involves mixing the patient's white blood cells withan extract of specific food and then measuring the cells in differentways for evidence of some form of change. There are a high number offalse positive and false negative reactions and the American Academy ofAllergy concluded that there was no evidence that the test was effectivefor the diagnosis of food or inhalant allergy.

6. Hair Analysis

Another form of testing is hair analysis. The hair can be analysed forthe presence of toxic metals such as Lead, Mercury and Cadmium or lowlevels of Selenium, Zinc, Chromium, Manganese and Magnesium. Heavy metalpoisoning is well recognised and documented in forensic science and hairanalysis can indicate exposure to metals. However, hair analysis as ameans to diagnosing allergy, through methods such as “dowsing”, havenever been validated.

7. Applied Kinesiology

Samples of food are placed under the tongue or held in a glass containerin the hand of the patient. The patient is then asked to push his freearm against that of the examiner. If an allergic response is detectedthis manifests as a reduced muscle response and the patient experiencesdifficulty in raising his arm. This technique fails to with stand upagainst a double blind study.

Furthermore, there are various in vitro methods for diagnosing allergieswhich detect the presence of IgE or IgG antibodies in the blood of apatient:

8. Conventional methods as ELISA or Western blots are used to detectantibodies (IgE) present in serum of a patient with the help of(recombinant) allergens.

9. Radioallergosorbent Test (RAST®):

In this procedure a specific allergen is coupled to a paper disk;immunospecific IgE, if present in the test (patient's) serum will bindto the disk; detection is effected by radiolabeled anti-IgE. Differentscoring systems comparing test results with the absolute binding of anegative control are in use. Commonly, the modified RAST® procedure isfollowed with overnight incubation and results in higher sensitivity.

10. RAST based quantitative IgE inhibition experiments in which allergenextract is dotted on nitro-cellulose strips. Aliquots of sera arepreincubated with a mixture of specific recombinant allergens afterwhich this mixture is incubated on the nitro-cellulose strips. Bound IgEis detected with anti human IgE antibodies. The percentage inhibition ofIgE binding the natural extracts after preabsorption with recombinantallergens is calculated in a last step.

11. Cellular antigens stimulation test (CAST) according to which patientbasophilic cells are stimulated with interleukin-3 and allergen followedby measurement in an ELISA of de novo generated and releasedsulfidoleukotriene (SLT).

12. UniCAP: Recombinant allergens are immobilized on a solid phasestructure after which the binding of antibodies which are present in theserum of a patient to the allergens is detected.

However, these methods require a multitude of individual experimentalsteps in order to test a larger number (e.g. 100) different allergens.Furthermore, these above mentioned tests do not show a sufficiently highsensitivity and reliability and are very time consuming. Also theamounts of sample (e.g. serum of a patient) as well as purified,possibly recombinant, costly allergens which are needed to carry outthese tests are large.

The U.S. Pat. No. 4,444,879 relates to methods and apparatus forimmnoassassays to determine total immunoglobulin and IgE. Here allergensare extracted and immobilized in polymer coated wells of amicrotiterplate. The sample to be analyzed is then added to the well anda conventional immuno assay is carried out.

In the EP 0 556 745 A1 a method for the detection of anti-wheat-proteinIgE antibodies in body fluids is described whereby a wheat allergenextract is immobilized onto a solid phase which is for examplechromatographic or capillary material or fibre, glass, nylon, celluloseor derivatives thereof in form of glass beads, microparticles, sheets orwells of a microtiterplate. Also here a conventional immunoassay iscarried out in order to detect antibodies in a sample of a patient.

The U.S. Pat. No. 3,720,760 relates to a method for analyzing body fluidfor immunoglobulins. Also here allergen containing extracts which arecommercially available are attached to water insoluble polymer afterwhich a conventional immuno assays is carried out.

The U.S. Pat. No. 4,849,337 relates also to a method for identifyingallergen specific IgE levels in a patient serum by conjugating the IgEin the serum with allergens adhering to an insoluble support. Here againthe allergen may be an extract or an allergen derived directly frompolens, dusts, animals, etc.

These conventional immuno assay methods, however, are not useful to testa patient with respect to the presence of allergies since the amount ofdifferent allergies has mounted to over a few hundred and is increasingsteadily. The time and work necessary to analyze a patient's serum forall possible end rare allergies is too much and will not be carried outin laboratories where a few hundred patient's samples must be analyzeddaily.

The object of the present invention is therefore to provide a method forthe detection of an immunoglobulin which binds to an allergen in asample which does not show the above mentioned drawbacks and which canbe carried out in a short time with only a small amount of sample andallergens, which is highly reliable and sensitive and most importantlyallows the detection of a practically unlimited number of allergens inone single assay.

A further object of the present invention is to provide a method for invitro diagnosis of allergies in a patient without the above mentioneddrawbacks.

The above mentioned method is characterized in that one or moreallergens are immobilized on a microarray chip after which the sample isincubated with the immobilized allergens so that immunoglobulins whichare specific for the allergens bind to the specific allergen after whichthe immunoglobulins which are bound to the specific immobilizedallergens are detected.

Microarrays have been adopted recently for a number of DNA manipulatingtechniques that are established in the scientific community since long.These DNA chips have become available in a number of different formatsand will eventually change ways of designing experiments in the ordinarylaboratory work. In vitro DNA diagnosis has become less time-consumingand labor-spending since this novel technology allows assay complexityin a high-throughput format. Consequently, in the area of proteomeresearch, classic solid phase substrates, such as microtiter plates,membrane filters and microscopic slides are being turned into thehigh-density microarray format. The new and versatile protein arraytechnology eventually allows high-throughput screening for geneexpression and molecular interactions (Walter et al., Curr OpinMicrobiol 2000 June;3(3):298-302; Emili & Cagney, Nat Biotechnol 2000April;18(4)393-7). Recently, the concept of protein arrays has beenadopted for its use in immunological assays.

A biochip is described as capable of supporting high-throughput (HT),multiplexed enzyme-linked immunosorbent assays (ELISAs). These biochipsmay consist for example of an optically flat glass plate containing 96wells formed by an enclosing hydrophobic Teflon mask, however, alsoother materials and forms of biochips are known and used. Experimentsdemonstrate that specific multiplex detection of protein antigensarrayed on a glass substrate is feasible. Further application of thisnew high-throughput screening (HTS) format include direct cellularprotein expression profiling, multiplexed assays for detection ofinfectious agents and cancer diagnostics (Mendoza L G et al,Biotechniques 1999 October;27(4):778-80).

However, in these known microarray chip methods testing proteins theantibodies are immobilized to the microarray chip. Surprisingly, it hasbeen found that it is possible not only to bind the antibodies to themicroarray chip but even allergens which are the antigens correspondingto specific antibodies, in particular IgE and IgG, respectively. Thisfact is particularly surprising since functional allergens show aparticular secondary structure which may be modified when bound in suchhigh density to a solid phase as the microarray chip. This modificationof the structure of the allergen would interfere with theantibody-allergen binding which would lead to false negative results.Antibodies, fragments of antibodies or peptides are relatively small andshow high stability, therefore antibodies show a greater stability insolution relative to their cognate antigens. However when immobilized toa solid support even in the case of antibodies only a small percentageremain intact and active.

The article by Haab et al. (Genom Biology 2000, I (6): pre-print0001,1-0001,22) which was received on 9 Nov. 2000 relates to proteinmicroarrays for detection and quantitation of proteins and antibodies insolutions. The results of the analysis carried out according to thispublication show that only 50% of the arrayed antigenes and 20% of thearrayed antibodies provided specific and accurate measurements of thecognate allergens.

Moreover, diversity of possible allergens is of course much higher thanantibody diversities with respect to handling the proteins, especiallyfor immobilizing and staining such a series of structurally diverseallergens. However, it has been surprisingly shown that allergensimmobilized on a microarray chip show high reliability and sensitivityin a method for the detection of immunoglobulins in a sample.

This method allows to carry out an assay for a multitude ofimmunoglobulins which bind to a specific allergen in one experimentalstep which assay can also be atomised: More than 100 different allergenscan be tested without having to carry out a multitude of individualexperimental steps as when performed in a conventional microtiter form.Furthermore, this assay shows a high degree of sensitivity andreliability. Also, the test results of this method can be obtainedwithin a short period of time, e.g. about three hours, shortening theassay time when compared to conventional RAST or ELISA tests.

Furthermore, the microarray assay is highly reproducible when performingrepetitive experiments with chips containing samples from differentpersons, e.g. by a microtiter plate-like mask whereby every wellcomprises a number of spots of different allergens and a sample of oneperson is added per well. A further advantage of this method accordingto the present invention is due to the fact that a large number ofdifferent probes occupies a relatively small area providing a highdensity. The small surface area of the array permits extremely uniformbinding conditions (temperature regulation, salt content, etc.) whilethe extremely large number of probes allows massively parallelprocessing of hybridizations. Because the high density arrays containsuch a large number of probes it is possible to provide numerouscontrols including, for example, controls for variations or mutations ina particular allergen, controls for overall hybridization conditions,controls for sample preparation conditions, and mismatch controls fornon-specific binding or cross hybridization.

Further, the assay requires only a minimal fraction of material,(allergen as well as a sample) when compared to conventional testsystems. This means that with an equal amount of starting material amuch greater number of test kits can be manufactured when using themicroarray form and a smaller amount of sample can be used to test for alarger amount of immunoglobulins which bind to allergens. Also, in smallvolumes, binding may proceed very rapidly.

Natural intact “immunoglobulins” or antibodies comprise a generallyY-shaped tetrameric molecule having an antigen binding-site at the endof each upper arm. An antigen binding site consists of the variabledomain of a heavy chain associated with the variable domain of a lightchain. More specifically, the antigen binding site of an antibody isessentially formed by the 3 CDRs (complementarity determining regions)of the variable domain of a heavy chain (V.sub.H) and the 3 CDRs of thevariable domain of the light chain (V.sub.L).

Generally the term “antigen” refers to a substance capable of elicitingan immune response and ordinarily this is also the substance used fordetection of the corresponding antibodies by one of the many in vitroand in vivo immunological procedures available for the demonstration ofantigen-antibody interactions.

Similarly, the term “allergen” is used to denote an antigen having thecapacity to induce and combine with specific (i.e., IgE) antibodieswhich are responsible for common allergies; however, this latterdefinition does not exclude the possibility that allergens may alsoinduce reactive antibodies, which may include immunoglobulins of classesother than IgE.

“Immobilizing” in the context of the proteins or peptides refers to thebinding or attaching of the protein/peptide to solid supports byconventional means, with or without an additional spacer between thesolid support and the allergen. Immobilizing peptides/proteins to asupport is well known in the art; in the scope of the present inventionany immobilization is comprised, e.g. covalent, non-covalent, inparticular by hydrophobic interactions with for example membranes andsynthetic surfaces, respectively, etc.

A “microarray” is an array of features (e.g. “spots”) having a densityof discrete features of at least about 16/cm², preferably at least about64/cm², still preferred about 96/cm² and most preferred at least about1000/cm². The features in a microarray have typical dimensions, e.g.,diameters, in the range of between about 10-2000 μm, preferably about50-500 μm, still preferred about 150-250 μm, and are separated fromother features in the array by about the same distance. Therefore, amicroarray according to the present invention useable for routine massscreening may have at least 500, preferably at least 1000 individualspots comprising allergens, standards and controls.

The “chip” according to the present invention may be of virtually anyshape, e.g. a slide or a well of a microtiter plate, or even a multitudeof surfaces although a planar array surface is preferred.

The detection step can be carried out with any conventional method knownby the person skilled in the art e.g. by physical, enzymatic, chemicalreaction, etc.

According to a preferred embodiment of the present invention IgEs aredetected as immunoglobulins. IgE is known as a substance that causesallergic reactions and is only produced when a person becomes sensitiveto a substance, an allergy. Therefore, in order to test if the sample isfrom a patient who is allergic to the specific allergen the sample istested for IgE as immunoglobulins. The higher the amount of IgE in thesample the more likely the patient the sample is taken from is allergicto the specific allergen.

Preferably IgG are detected as immunoglobulins. IgG is known to bepresent in a sample of a patient who is allergic to food, therefore thedetection of IgG can be used as an indication for food intolerance.

Advantageously one or more purified allergens are immobilized on themicroarray chip. These—single—allergens are for example purified from anallergen extract, e.g. from a specific food or plants. Of course, one ormore allergens of a specific species can be analysed at once.

The methods of purification are known to the person skilled in the art,e.g. chromatographic purification, purification by mass separation,purification by specifically binding the allergen to a solid phase, e.g.over an antibody, etc. Application of highly purified allergens for massproduction of allergy testing has not yet been proposed.

“Purified” allergens relate—to the contrary of extracts—to singleallergens which can be for example native, recombinant or syntheticallyproduced allergens. These purified allergens show a number of importantadvantages over allergen extract when immobilized to a solid support:Due to the mixture of various proteins which are present in any extractthe concentration of allergens to be tested is very low compared topurified single allergen preparations. Purified single allergens cantherefore be immobilized to the microarray in a very high concentration.Due to the exact and defined molecules present in a preparationcomprising purified allergens only defined allergens will be immobilizedto the micro array. Therefore, the microarray and the method fordetecting antibodies with the help of purified allergens can bestandardized and subjected to precise quality controls.

Furthermore, due to the high concentration of purified allergens in thecomposition the allergens are immobilized at a higher density on themicroarray and therefore any detection method with the help of such amicroarray is more sensitive than corresponding microarrays withextracts comprising allergens. Another advantage of purified singleallergens over allergen extracts lies in the fact that the purifiedallergens immobilized are exactly defined. To the contrary of purifiedallergens allergen extracts comprise for example two, three or moredifferent allergens of one organism or object. For example, in the caseof an apple, an extract will comprise a mixture a different appleallergens, whereas the inventive composition will comprise one of thepurified apple allergens or—depending on the use—a defined mixture oftwo or more apple or other allergens.

A further advantage of purified allergens with respect to non-purifiedallergens for example present in extract lies in the fact that due tothe additional impurities present in the extract the extract is notstable for more than a certain amount of time and can for example gomouldy. This will, however, influence the detection of allergies sincealso allergies against mould exists in which case a false positiveresult would occur.

Furthermore, due to the presence of proteases in the extract theextracts are instable which is not the case with purified allergens.

Also, due to the at present existing therapies with the help of purifiedallergens a diagnosis with the same purified allergen components wouldbe a major advantage over diagnosis with unpurified allergens. Suchdiagnoses are particularly advantageous in order to follow the therapywith purified allergens and to test the individual reaction to aspecific therapy and thereby to provide a “patient tailored” therapy.

For the above reasons a method for the detection of an immunoglobulinwhich binds to the allergen in a sample whereby one or more purifiedallergens are immobilized to the microarray chip is particularlyadvantageous.

Preferably one or more recombinant allergens are immobilized on themicroarray chip. Over the last few years a number of recombinantallergens have been produced. The production of recombinant allergens isalso in principle known state of the art but had little impact onroutine allergy testing. By using recombinant allergens it is possibleto provide a great number of one or more specific allergens andtherefore to optimize sensitivity of the test. It is furthermorepossible to modify the recombinant allergens specifically in order toproduce any allergen mutations to detect for specific immunoglobulins.Furthermore, making use of the major allergens as recombinant proteinsprovides an alternative to the extract based tests with respect to assaystability as well as to diagnostic standardisation. The above mentionedadvantages of purified allergens over the unpurified (extract) allergensapply even more for recombinant allergens than for purified nativeallergens: Recombinant allergens can be even more specifically designedthan the purified native allergens and therefore it is possible tooptimize affinity and specifity of a test with recombinant allergens.Recombinant allergens are also better to standardize with respect totheir production method. Moreover, differences between production lotsare smaller for recombinant allergens than for allergens derived fromnatural sources. It has been surprisingly shown with the presentinvention that the use of these recombinant allergens in the routine invitro diagnostic tests according to the present invention is superior toconventional extract based test systems. In contrast to the routineserial testing according to the state of the art, the present allergentesting using also recombinant allergens brings a completely new qualitywith respect to standardisation, controllability, sensitivity,reproducibility, the ability to test for diagnostic relevantinformation, etc. for standard allergen testing.

Recombinant allergens are for example those described in thepublications by Chapman et al. Allergy, 52:374-379 (1997), Laffer et al.J Allergy Clin Immunol Vol 98 Number 2 652-658 (1996), Müller et al.Clinical and Experimental Allergy Vol 27 9. 915-920 (1997), Niederbergeret al. J Allergy Clin Immunol Vol 102 Number 4, Part 1 579-591 (1998),Menz et al. Clinical and Experimental Allergy Vol 26, 50-60 (1996) whichare incorporated herein by reference. Further examples of (recombinant)allergens are but not limited to rBetv1, rBetv2, rBetv4, rJunO2, Cass1,rPhlp1, rPhlp2, rPhlp4, rPhlp5a, rPhlp6, rPhlp7, rPhlp11, rPhlp12,rParj2, Artv1a, Mugwort profilin, Apig1, Apig1.0201, Dauc1.2, rArah2,rArah5, Mald1, Mald2, rPena1, recCarp, rDerp1, rDerp2.0101, rDerp2,rDerp2b, rDerp5, rDerp5a, rDerp7, rDerp8, rDerp10, rTyrp2, rLepd2.01,rLepd13, rEurm2.0101, rFeld1, rFeld1a, rBosd2, a representative allergenfrom Cat, a representative allergen from Dog, a representative allergenfrom BSA, a representative allergen from Mouse, a representativeallergen from Rat, a representative allergen from Pig, a representativeallergen from Sheep, a representative allergen from Chicken, arepresentative allergen from Rabbit, a representative allergen fromHamster, a representative allergen from Horse, a representative allergenfrom Pigeon, a representative allergen from Guineaalbumin, HSA, a-NAC,rPenc3, Penn13, rPenc19a, rPenc19b, rAspf1, rAspf1a, rAspf3, rAspf3a,rAspf4, rAspf4a, rAspf6, rAspf6a, rAspf7, rAspf8, rAlta1, rAlta2,rMalf1, rMalf5, rMalf6, rMalf7, rMalf8, rMalf9, rHevb1, rHevb1a, rHevb3,rHevb8, rHevb9, rHevb10, rHevb11, rAK, rBlag2, rBlag4, rBlag5,Phospholipase A, Hyaluronidase, rVesv5, rVesg5.

According to a further advantageous embodiment one or more syntheticallyproduced allergens are immobilized on the microarray chip. Here againthe same advantages over allergen extract and also over purified nativeallergens apply as mentioned above for recombinant allergens. The methodfor synthetically producing peptides or proteins is well known in thestate of the art; by synthetically producing allergens it is possible toprovide highly purified allergens in a great number and at low costssince such production methods are highly automatized. Furthermore, theexact sequence of any allergen can be provided with or withoutmodifications, which increases the sensitivity and reliability of themethod for detecting the immunoglobulins.

It is also possible to use only the allergenic determinant or theallergenic domain of a specific allergen in the test according to thepresent invention. This may eliminate the risks and drawbacks of workingwith large proteins, because shorter peptidic structures are easier tohandle for routine production of biochips. This applies to all purifiednative, recombinant and synthetic allergens. This would further allow todetect single peptide epitopes which would still improve the diagnostictest and therapies in particular with respect to specificity.

It is of particular advantage to provide the native form of the allergenas well as a synthetic or recombinant form of the allergen on the samechip. Whereas it is preferred to use mainly recombinant or syntheticallergens the native form of an allergen may be provided at least as acontrol or additional quality information on the chip. A preferred chipaccording to the present invention therefore comprises a native and asynthetic or recombinant form of at least one allergen. This providesalso for improved quality control and standardisation of differentbatches of allergens.

Preferably one or more haptens are immobilized as allergens on themicroarray chip. A “hapten” is a low molecular weight (typicallyweighing less than about 7000 Daltons) substance that is generallyincapable of causing, by itself, a significant production of antibodiesupon administration to an animal body, including a human body. This canoccur because a hapten is too small to be recognized by the body'simmune system. However, when a hapten is coupled to a larger, carriermolecule, the hapten can acquire antigenic properties. In other words,binding the hapten to the carrier molecule (to make an analyte-carriermolecule combination) permits the bound hapten to be recognized by ananimal's immune system. An immunoprecipitation reaction can take placebetween the hapten (coupled to the carrier molecule), and an antibody tothe hapten. By providing haptens which are immobilized to the microarraychip a higher density can be achieved on the chip.

It is of course possible to combine these above mentioned differentforms of allergens, e.g. to use at the same time recombinant and(purified) native allergens. The combination of these different formsallows to compare them and carry out a control of the e.g. recombinantallergens but also as a control for the various native lots of theallergens which may vary due to differences in biological sources,methods of preparation, purity, etc.

For a highly efficient test it is preferable to use allergens which showoptimal features, e.g. a high binding capacity. However, the use of lessor differently active antigens is preferred for the detection ofinefficiently binding allergen-variants, e.g. for the use inhyposensitization therapies.

According to a preferred embodiment of the present invention theallergens are immobilized on a 10 to 2000 μm diameter, preferably 50-500μm diameter, still preferred 150-250 μm diameter, spot on the microarraychip. Since the spots have a small diameter, it is possible toimmobilize a great amount of different allergens and variousconcentrations of specific allergens on separate spots of the microarraychip, thus allowing to provide one microarray chip which can inone—automatized—step be used to analyse a big number of allergens orallergen concentrations.

Preferably the allergens are immobilized on a solid support, preferablya glass carrier, synthetic carrier, silicon wafer and membrane,respectively. Such chips can be produced for example according tomethods described in Ge, H. (2000), Nucleic Acids Research, 28, e3(i-vii); Qian W. et al. (2000), Clinical Chemistry, 46 (1456-1463);MacBeath G. et al. (2000), Science, 289, (1760-1763), which aredisclosed herein by reference. Each of these materials shows specificcharacteristics and advantages which are well-known to the personskilled in the art. Therefore, the material for the microarray chip willbe chosen according to the allergen which is to be tested, the methodfor the detection of bound immunoglobulins, the used buffers.Furthermore, the choice of material is also a financial question.

Preferably the microarray chip is chemically modified, preferably byaminoreactive and carboxyreactive modification, respectively. Thisallows to precisely determine the way the allergen is bound to themicroarray chip.

Advantageously the allergens are covalently bound to the microarraychip. This provides a stable binding of the allergens to the microarraychip, thereby providing a method which is particularly reliable.

According to another advantageous embodiment of the present inventionthe immunoglobulins are detected in blood serum as the sample. Inpatients who are allergic to an allergen immunoglobulins are producedmainly in the blood serum, so that analyzing the blood serum provides areliable method for detecting immunoglobulins. Furthermore, blood serumcan easily be collected from the patient in a very simple way and thecollection of the sample can be carried out even at the home of thepatient.

Preferably the blood serum is diluted 1:1-1:15, preferably 1:5. Thedilution can be carried out with any suitable solution, e.g. 1×TBST (10mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% TWEEN 20)

Advantageously the sample is incubated 1 min. to 24 hours, preferably1-2 hours, with the allergens. Of course, it is possible to incubate thesample with the allergens even over a period of days. However, this doesnot result in an increase in signal intensity or specificity. Ingeneral, an incubation time of 1 hour is sufficient for a reliable andsensitive result.

Furthermore, the sample is preferably incubated at a temperature between0 and 60° C., preferably at 37° C., with the allergens. Incubations atlower temperatures do not result in an increase of signal, but theyrather lead to an increase in unspecific binding and background noise.An incubation at 37° C. has shown to provide exact and reliable resultsfor allergen test in humans.

Advantageously the bound immunoglobulin is detected with at least onelabelled specific anti-immunoglobulin antibody. As used herein, the term“antibody” refers to intact molecules as well as fragments thereof, suchas Fa, F(ab).sub.2, and Fv, which are capable of binding the epitopicdeterminant. Antibodies can be prepared using intact polypeptides orfragments containing small peptides of interest as the immunisingantigen. The polypeptide or peptide used to immunise an animal can bederived from the transition of RNA or synthesized chemically, and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunise the animal(e.g., a mouse, a rat, or a rabbit).

In the scope of the present invention the term antibody refers tomonoclonal or polyclonal antibodies whereby monoclonal antibodies may beprepared using any technique which provides for the production ofantibody molecules by continues celllines in culture. These include butare not limited to the hybridoma technique, the human B-cell hybridomatechnique and the IBV hybridoma technique. Furthermore, chimericantibodies may be produced, single chain antibodies as well assynthetically produced antibodies.

The antibody may be labelled according to any known method which allowsto qualify and preferably quantify the bound antibody. Detectable labelssuitable for use in the present invention include any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Useful labels include biotin forstaining with labelled streptavidin conjugate, magnetic beads,fluorescent dyes (e.g. fluorescein, texas red, rhodamine, greenfluorescent protein, and the like), radiolabels (e.g. ³H, ¹²⁵I, ³⁵S, ¹⁴Cor ³²P), enzymes (e.g. horse radish peroxidase, alkaline phosphatase andothers commonly used in ELISA), and calorimetric labels such ascolloidal gold or coloured glass or plastic (e.g. polystyrene,polypropylene latex, etc.) beads.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent labels may be detected usinga photodetector to detect emitted light. Enzymatic labels are typicallydetected by providing the enzyme with a substrate and detecting thereaction product produced by the action of the enzyme on the substrate,and colorimetric labels are detected by simply visualising the colouredlabel.

Preferably the bound immunoglobulins are detected with at least onefluorescence and radioactive, respectively, labelled specificanti-immunoglobulin antibody. These are classic methods forqualitatively and quantitatively analyzing bound antibody which are veryspecific and reliable.

Other advantageous detection systems for micro arrays are for examplethose described in Schult K. et al. (1999), Anal Chem, 71 (5430-5435);Vo-Dinh T. et al. (1999), Anal Chem, 71 (358-363); Brignac S J Jr. etal. (1999), IEEE Eng Med Biol Mag, 18 (120-122); Otamiri M. et al.(1999), Int J Biol Macromol, 26 (263-268); Wright, G L Jr. et al.(2000), Prostate Cancer and Prostatic Diseases, 2 (264-276); Nelson R W.et al. (2000), Electrophoresis 21 (1155-1163); Rich R L. et al. (2000),Curr Opin Biotechnol 11 (54-61); Chen J J. et al. (1998), Genomics, 51(313-324), which publications are enclosed herein by reference.

Advantageously one or more indoor allergens are immobilized asallergens. These may be but are not limited to Mites, Tyr. put, Lep.dest. or .mayrei, Felis, Bos, Albumine, Pen. cit., Pen. not., Asp.fumigatus, Alt. alt., Malassezia furfur, Latex, Plodia, Blatella.

According to a further preferred embodiment one or more outdoorallergens are immobilized as allergens. These may be but are not limitedto Betula, Juniperus, Phleum, Parietaria judicea.

Further one ore more food allergens may be immobilized as allergens.Examples are sellerie, karott, peanut, apple, shrimp, fish.

Further one ore more venom allergies are immobilized as allergens. Thesemay be but are not limited to Bee or Wasp.

Also one or more auto-allergens may be immobilized as allergens. Suchauto-allergens may be e.g. liver membrane antigens, ssDNA antigens,antigens in or on skeleton muscle cells, etc.

A further aspect of the present invention relates to a method for invitro diagnosis of allergies in a patient, whereby a serum sample istaken from the patient after which the sample is analysed forimmunoglobulins which bind to allergens according to an above mentionedmethod according to the present invention, whereby a microarray chip isused on which at least 10, preferably at least 50, still preferred atleast 90, different allergens are immobilized, after which a positivereaction between the sample and the immobilized allergens is diagnosedas an allergy. The above mentioned definitions and advantages relatealso to this method for in vitro diagnosis of allergies compared to theknown methods for diagnosing allergies. The present method shows theadvantage that a big amount of allergies can be simultaneously diagnosedwith only one sample since all the allergens which are to be tested areimmobilized on one single microarray chip. Every step is thereforecarried out only on one chip, whereby the chip further may comprisespots with no immobilized allergens and therefore allowing simultaneousnegative detection. The amount of allergens to be tested is not limitedand it is of course possible to provide two or more microarray chips.

A further aspect of the present invention relates to a microarray chipon which one or more allergens are immobilized. Also in this aspect theabove mentioned definitions and advantages are applied.

Preferably the allergens are immobilized on a 100 to 500 μm diameter,preferably to 200-300 μm diameter, spot.

Still preferred the microarray chip is a glass carrier, syntheticcarrier, silicon wafer and a membrane, respectively.

Advantageously the microarray chip is chemically modified, preferably byaminoreactive and carboxyreactive modification, respectively.

Advantageously the allergens are covalently bound to the microarraychip.

A further aspect according to the present invention relates to a kitwhich comprises a microarray chip according to the present invention asmentioned above and a first reagent comprising at least oneimmunoglobulin detecting reagent, preferably an anti-immunoglobulinantibody, preferably in a known concentration, and possibly a secondreagent as a positive sample comprising at least one immunoglobulinwhich binds to an allergen. The first reagent comprising the at leastone immunoglobulin detecting reagent, preferably an anti-immunoglobulinantibody may be used for the detection of bound immunoglobulin in whichcase the anti-immunoglobulin antibody is preferably labelled asmentioned above. Preferably an antibody is present in the first reagentin a known concentration thereby allowing to provide for reproducibleresults. Furthermore, the kit preferably comprises a second reagent as apositive sample comprising at least one immunoglobulin which binds to anallergen. Also here it is preferable that the immunoglobulin is presentin a known concentration in the second reagent thereby allowing toquantify the immunoglobulin present in the sample by comparing theresults of the sample of the patient with the results of positive sample(the second reagent).

Preferably the kit is provided for carrying out a method according tothe present invention as mentioned above. For this the first and secondreagents may be designed in order to detect one specific immunoglobulinwhich binds to a specific allergen or one specific allergy in a patient.However, the kit may also be designed to detect a variety ofimmunoglobulins which bind to specific allergens or to diagnose avariety of allergies in a patient.

The present invention is now described in more detail with the followingexamples and figures to which it is of course not limited.

FIG. 1 shows a layout of an allergen microarray;

FIG. 2 shows a scan image of an allergen array assayed with serum froman allergic person;

FIG. 3 a-3 c show a Scatterplot Test from a triple assay;

FIG. 4 a-4 c show the % fluorescence measured for immobilised extractand immobilised recombinant allergens.

EXAMPLE 1 Allergen Spotting

Allergens were arrayed using a GMS 417 spotter from GeneticMicrosystems. The proteins were spotted on derivatized glass slides.Individual allergens were spotted at one hit per dot as triplicates. Thedots (features) showed a diameter of about 200 μm.

The allergens were assembled in functional groups as follows:

(A) Outdoor (I. Trees [1=rBetv1, 2=rBetv2, 3=rBetv4, 4=rJunO2, 5=Cass1],II. Grasses [6=rPhlp2, 7=rPhlp4, 8=rPhlp5a, 9=rPhlp1, 10=rPhlp6,11=rPhlp7, 12=rPhlp11, 13=rPhlp12], III. Weeds [14=rParj2, 15=Artv1a,16=Mugwort profilin]),

(C) Food Allergens (X. Vegetables [17=Apig1, 18=Apig1.0201, 19=Dauc1.2,20=rArah2, 21=rArah5], XI. Fruits [22=Mald1, 23=Mald2], XII. Shrimps[24=rPena1], XIII. Fish [25=recCarp]),

(B) Indoor (IV. Mites [26=rDerp1, 27=rDerp2.0101, 28=rDerp2, 29=rDerp2b,30=rDerp5, 31=rDerp5a, 32=rDerp7, 33=rDerp8, 34=rDerp10, 35=rTyrp2], V.Animals [36=rLepd2.01, 37=rLepd13, 38=rEurm2.0101, 39=rFeld1,40=rFeld1a, 41=rBosd2, 42=a representative allergen from cat, 43=arepresentative allergen from dog, 44=BSA, 45=a representative allergenfrom mouse, 46=a representative allergen from rat, 47=a representativeallergen from pig, 48=a representative allergen from sheep, 49=arepresentative allergen from chicken, 50=a representative allergen fromrabbit, 51=a representative allergen from hamster, 52=a representativeallergen from horse, 53=a representative allergen from pigeon,54=guineaalbumin], VI. Moulds [55=rPenc3, 56=rPenc19a, 57=rPenc19b,58=Penn13, 59=rAspf1, 60=rAspf3, 61=rAspf4, 62=rAspf6, 63=rAspf1a,64=rAspf3a, 65=rAspf4a, 66=rAspf6a, 67=rAspf7, 68=rAspf8, 69=rAlta1,70=rAlta2], VII. Yeast [71=rMalf7, 72=rMalf1, 73=rMalf5, 74=rMalf6,75=rMalf8, 76=rMalf9], VIII. Latex [77=rHevb1, 78=rHevb1a, 79=rHevb3,80=rHevb8, 81=rHevb9, 82=rHevb10, 83=rHevb11], IX. Insects [84=rAK,85=rBlag2, 86=rBlag4, 87=rBlag5]),

(D) Venoms (XIV. Bee [88=Ag5, 89=Phospholipase A, 90=Hyaluronidase,91=rVesv5, 92=rVesg5], XV. Wasp [93=Ag5]),

(E) Auto-allergens (94=HSA, 95=a-NAC)

Additionally, buffer dots were interspersed between the allergensubgroups serving as a background control, marked as “X”, “ab” is thelabelled antibody. (FIG. 1: 1864×1182 pixels, 1.86×1.18 cm, 1000 pixelsper cm, pixel depth/colours 8/256, 1 pixel corresponding to 10 μm).

100 μl aliquots of the allergens were divided into the wells of a 96well microtiter plate at a concentration of ˜200 ng/μl in spottingbuffer (300 mM Sodium-phosphate pH 8.5). The optimal concentration forimmobilization of the allergens was calculated from titrationexperiments with several allergens in different buffer solutions as wellas on different slides. The proteins assayed displayed a saturationbehaviour at concentrations equal to or larger than 100 ng/μl as becameevident from a constantly strong white signal when scanning with a GMSscanner. At this concentration the binding behaviour of the allergenswas independent of the composition of the storage as well as thespotting buffer.

EXAMPLE 2 SOPHIA (Solid Phase Immunosorbent Assay)

Following over night incubation slides either purchased from CELAssociates (aldehyde slides) or prepared in-house were washed in 1×TBST(10 mM Tris pH 8.0/150 mM NaCl/0.5% Tween 20) under vigorous shaking ina Falcon tube at ambient temperature. As a blocking step, the slideswere transferred to a 1×TBST solution containing 0.01% BSA for 2 hoursat ambient temperature. Successively, slides were washed in 1×TBST for15 min. and rinsed in distilled water briefly.

The allergen array was incubated with diluted serum (1:5 in 1×TBST) for(at least) 60 min. at 37° C with shaking. Various degrees of serumdilution have been tested, ranging from 1:1-1:15. Usually a dilution of1:5 gave the best results. 30 μl of diluted serum was added to theslides in Press Seal Chambers purchased from SIGMA Technologies. Thechambers were used as according to the manufacturers protocol. Variousincubation times for the serum ranging from 60 min. to over night wereassayed. However, an extended incubation with serum did not result in anincrease in signal intensity or specificity. Following the incubationwith serum slides were washed in 1×TBST (15 min., ambient temperature)with shaking.

Five different serums of allergic patients that have been examined usingconventional diagnostic tests (skin prick test, RAST, ELISA) and a serumof a non-atopic patient were chosen adequate for a benchmark test of theallergen array. The individual sera were assayed at least twice ondifferent batch produced allergen arrays.

Fluorescence-labelled α IgE antibody labelled with AlexaFluorfluorescence dye according to the manufacturer's protocol (MolecularProbes) was added to the allergen array in a solution containing 0.01%BSA/1×TBST and incubated for 60 min. at ambient temperature. Workingdilutions for the antibody ranged from 1:1000-1:5000 depending on thetime and efficiency of labelling. Following the immunoassay, slides werewashed with 1×TBST (15 min., ambient temperature, shaking) and brieflyrinsed with distilled water.

Following the immunosorbent assay, the slides were evaluated with a GMStwo-colour scanner. Generally, signal intensities varied only slightlybetween different assays and different slides. However, the backgroundvalues differed depending on the type of slides used. An example of ascanned image of an allergen array assayed with serum of a patientsuffering of allergy is depicted in FIG. 2. This patient shows a strongreaction with Phleum, Mite, Felis and bee (cf. FIG. 1). No backgroundsignals stemming from an unspecific interaction with buffer dots orauto-allergens were observed. Following the complete evaluation of theallergic patient's sera the results were compared with data preliminaryobtained for the identical sera using RAST tests. The data achieved withthe method according to the present invention were in good agreementwith those data sets available

EXAMPLE 3 Reproducibility Test Assaying Serum of Patient C

Serum of patient C was assayed three times on individually preparedallergen-microarrays. The experimental procedure was as essentiallydescribed before.

Following the assay, the slides were scanned using identical hardwaresettings. Data analysis was performed using the GenePix softwarepackage. The calculated mean values of the signal intensities for eachallergen triplicate were compared after three repeats of the experiment.

Only values corresponding to signals that were at least 1.5× higher thanthe mean value of the buffer spot signals were chosen for the finalanalysis. The mean value, standard deviation and percentage of standarddeviation for the relevant signals are depicted in table 1. TABLE 1Allergen Test 1 Test 2 Test 3 Mean Value Standard Dev. % Mean rBet v 120731 21325 25060 22372.00 1916.11 8.56 rPhl p 2 17176 15529 1422715644.00 1206.67 7.71 rPhl p 4 36134 33511 29328 32991.00 2802.76 8.50rPhl p 5a 56600 53068 55594 55087.33 1485.77 2.70 rPhl p 6 56244 5135937625 48409.33 7882.14 16.28 rPhl p 12 6291 7003 12289 8527.67 2675.5031.37 rPar l 2 7552 7678 9444 8224.67 863.73 10.50 Art v 1a 6997 793111258 8728.67 1828.70 20.95 Mugwort profilin 7901 7669 9260 8276.67701.74 8.48 Mal d 1 9890 15295 8176 11120.33 3033.74 27.28 HSA 9868 873210708 9769.33 809.71 8.29 Sheepalbumin 9858 8295 10222 9458.33 835.928.84 Horsealbumin 9602 9061 10559 9740.67 619.37 6.36 rAsp f 1 (b) 1221610129 10781 11042.00 871.77 7.90 rMal f 5 13459 11018 12035 12170.671001.14 8.23 rAK 20680 14202 19978 18286.67 2902.48 15.87

The mean standard deviation calculated from the values obtained afterthree individual assays was 12.36%. This means that the chip-basedimmunological assay, questioning the presence of IgE in patients' sera,is highly reproducible. This is also evident when inspectingscatter-plots derived from the triple-assay 3, s. FIG. 3 a-3 c, whereinFIG. 3 a shows a Scatterplot Test 1 vs. Test 3, FIG. 3 b a ScatterplotTest 2 vs. Test 3 and FIG. 3 c a Scatterplot Test 1 vs. Test 2; Ashowing the allergens and FI the fluorescence intensity.

EXAMPLE 4 Comparison of Different Microarrays

In order to test for the optimum slide for the present invention,individually prepared slides were evaluated according to a number ofcriterion that are outlined below:

-   -   Production process: Slides that were prepared were evaluated        according to a number of parameters, such as hazardous chemical        requirement and production time.    -   Cost of production: Slides produced in house and commercially        available slides were compared according to their costs.    -   Binding capacity: Different slides were evaluated according to        the binding capacity of a fluorescent labelled protein.    -   Reproducibility: Serial experiments were performed with        differently pretreated slides and evaluated according to the        overall assay performance.    -   General Background: All slides were evaluated before and after        an allergy assay for a systematic surface background that might        diminish the signal to noise ratio.    -   Detection limit: All slides were evaluated for the minimal        protein concentration detectable per spot in an allergy        screening assay.    -   Serum tolerance: Generally, a patient's serum is a complex        mixture of proteins that often interferes negatively with a        microarray-based immunoassay with different batches of        patients's serum.    -   Blocking: All slides were evaluated for necessity of a blocking        step prior to the allergy assay.    -   Storage: All slides were evaluated for long-time storage after        an allergen chip has been produced.

The results of the above mentioned evaluation study are depicted intable 2: TABLE 2 Reactive Surface Chemistry/ Binding General DetectionAllergy Blocking/ Slide Type Production Cost Capacity SpecificityReproducibility Background Limit Screening Coupling Storage ProteoBind + ++ ++ ++ ++ ++ ++ ++ No − CEL (1) / ++ ++ ++ − − + ++ Yes ++3D-Link (2) / −− ++ ++ + + + − Yes − Superaldehyde (1) / −− + + ++ − + +Yes ++ Aminosilane (3) + ++ + + −− − + + No ++ Glyoxal (3) − + ++ ++ −−− + ++ Yes ++ EGS (3) − +/− ++ ++ −− − + + No − Sulfo KMUS (3) − +/−++ + / − / / No / Glutaraldehyde − +/− / / −− − / / / ++ (3)Photocrosslinker − +/− + + / − / / / / (3) PEI/EGS (3) − −− ++ + + + / // / FAST Slides (4) / −− + −− − −− −− − No ++ CAST Slides (4) / −− + + +−− −− −− Yes ++ Unmodified glass + ++ − / −− ++ −− / / /

Table 2. Depicted are the results of the evaluation study described inthe text. (++) means excellent result, (+) good, (−) minor, (−−) bad.(/) means that the slide has not been assayed for this particularcategory. (1) Slides containing a functional surface with aldehydegroups. (2) Slides containing an amine-reactive surface, the exactchemical properties of which are not known. (3) Slides produced in-housewith functional groups as mentioned in table 2. (4) Slides containing amembrane for immobilization of proteins. ProteoBind is the working titlefor the surface derivatisation adapted for an optimized performance ofin microarray-based allergy diagnosis and comprises(1-[3″-[-tri-methoxysilyl)propyl]-1′(4″-isothiocyanatophenyl) thiourea),which was prepared according to Chen et al. (Nucleic Acids Research,199, vol. 27, No. 2), which is incorporated herein by reference.

According to the evaluation study presented above ProteoBind wasselected as a superior surface derivatisation for the production ofallergen microarrays.

EXAMPLE 5 Comparison of the Detection of Immunglobulin in a Sample withImmobilized Recombinant Allergens and with Immobilized Extract

In order to test the sensitivity and diagnostic relevant information ofpurified recombinant allergens immobilized to a microarray and ofallergen extract immobilized to a microarray, specific allergens of onesource were compared to an allergen extract of the same source.

After immobilization of allergen compositions to a microarray differentsamples comprising said specific serum were added onto the mircroarrayand the fluorescence which corresponds to the amount of antibody boundto the allergens was measured. The results are shown in tables 3, 4 and5 as well as FIGS. 4 a, 4 b and 4 c, where “FL” stand for %fluorescence. From these results it is clear that in the case of therecombinant allergens, detection and quantification is much moresensitive than in the case of the allergen extracts. The fluorescenceintensity of a single recombinant antigene is clearly higher than thefluorescence intensity of the extract. Furthermore, in the case of theextract only the source of the extract can be tested and it is notpossible to test which specific antigene of the source is responsiblefor the allergy to the contrary of detection with recombinant allergens.

Therefore, the inventive method using single purified allergens, inparticular recombinant allergens are advantageous over extractscomprising allergens. TABLE 3

TABLE 4

TABLE 5

1-37. (canceled)
 38. A method for the detection of am immunoglobulin which binds to an allergen in a sample comprising: immobilizing one or more allergen on a microarray chip; incubating a sample with the microarray chip such that immunoglobulins which are specific for one or more of the one or more allergen, if any such immunoglobulins are present, bind to the one or more allergen; and detecting any immunoglobulins which are bound to the one or more allergen detected.
 39. The method of claim 38, wherein an immunoglobulin bound to the one or more allergen is detected.
 40. The method of claim 39, wherein the detected immunoglobulin is further defined as an IgE.
 41. The method of claim 39, wherein the detected immunoglobulin is further defined as an IgG.
 42. The method of claim 38, wherein the one or more allergen is further defined as a purified allergen.
 43. The method of claim 38, wherein the one or more allergen is further defined as a recombinant allergen.
 44. The method of claim 38, wherein the one or more allergen is further defined as a synthetically produced allergen.
 45. The method of claim 38, wherein the one or more allergen is further defined as a hapten.
 46. The method of claim 38, wherein the one or more allergen is immobilized on a 10 to 2000 μm diameter spot on the microarray chip.
 47. The method of claim 46, wherein the one or more allergen is immobilized on a 50 to 500 μm diameter spot on the microarray chip.
 48. The method of claim 47, wherein the one or more allergen is immobilized on a 150 to 250 μm diameter spot on the microarray chip.
 49. The method of claim 38, wherein the microarray chip is further defined as comprising a solid support.
 50. The method of claim 38, wherein the microarray chip is further defined as comprising a glass and synthetic carrier.
 51. The method of claim 38, wherein the microarray chip is further defined as comprising a silicon wafer.
 52. The method of claim 38, wherein the microarray chip is further defined as comprising a membrane.
 53. The method of claim 38, wherein the microarray chip is further defined as chemically modified.
 54. The method of claim 53, wherein the microarray chip has been modified by aminoreactive and carboxyreactive modification.
 55. The method of claim 38, wherein the at least one allergen is covalently bound to the microarray chip.
 56. The method of claim 38, wherein the sample is blood serum.
 57. The method of claim 56, wherein the blood serum is diluted 1:1-1:15.
 58. The method of claim 57, wherein the blood serum is diluted 1:5.
 59. The method of claim 38, wherein the sample is incubated with the at least one allergen for from 1 minute to 24 hours.
 60. The method of claim 59, wherein the sample is incubated with the at least one allergen for from 1 to 2 hours.
 61. The method of claim 38, wherein the sample is incubated at a temperature between 0 and 60° C.
 62. The method of claim 61, wherein the sample is incubated at a temperature of about 37° C.
 63. The method of claim 38, wherein any bound immunoglobulins are detected with at least one labeled, specific anti-immunoglobulin antibody.
 64. The method of claim 63, wherein any bound immunoglobulins are detected with at least one fluorescence labeled specific anti-immunoglobulin antibody.
 65. The method of claim 63, wherein any bound immunoglobulins are detected with at least one radioactive labeled specific anti-immunoglobulin antibody.
 66. The method of claim 38, wherein the one or more allergen is further defined as an indoor allergen.
 67. The method of claim 38, wherein the one or more allergen is further defined as an outdoor allergen.
 68. The method of claim 38, wherein the one or more allergen is further defined as a food allergen.
 69. The method of claim 38, wherein the one or more allergen is further defined as a venom allergen.
 70. The method of claim 38, wherein the one or more allergen is further defined as an auto-allergens allergen.
 71. The method of claim 38, further defined as a method for in vitro diagnosis of allergies in a patient, wherein: the sample is a serum sample taken from the patient; the sample is analyzed for immunoglobulins which bind to allergens immobilized on a microarray chip on which at least 10 different allergens are immobilized; and binding of at least one immunoglobulin from the sample to at least one of the immobilized allergens indicates an allergy in the patient.
 72. The method of claim 71, wherein the microarray chip comprises at least 50 different immobilized allergens.
 73. The method of claim 72, wherein the microarray chip comprises at least 90 different immobilized allergens.
 74. A microarray chip on which at least one allergen is immobilized.
 75. The microarray chip of claim 74, wherein the at least one allergen is immobilized on a 100 to 500 μm diameter spot.
 76. The microarray chip of claim 75, wherein the at least one allergen is immobilized on a 200 to 300 μm diameter spot.
 77. The microarray chip of claim 74, comprising a glass carrier.
 78. The microarray chip of claim 74, comprising a synthetic carrier.
 79. The microarray chip of claim 74, comprising a silicon wafer.
 80. The microarray chip of claim 74, comprising a membrane.
 81. The microarray chip of claim 74, further defined as a chemically modified microarray chip.
 82. The microarray chip of claim 81, further defined as chemically modified by aminoreactive and carboxyreactive modification.
 83. The microarray chip of claim 74, wherein the at least one allergen is covalently bound to the microarray chip.
 84. The microarray chip of claim 74, further defined as comprised in a kit.
 85. The microarray chip of claim 84, further defined as comprised in a kit comprising the microarray chip and at least one immunoglobulin detecting reagent.
 86. The microarray chip of claim 84, further defined as comprised in a kit for the detection of an immunoglobulin which binds to an allergen in a sample.
 87. A kit comprising a microarray chip on which at least one allergen is immobilized and a first reagent comprising at least one immunoglobulin detecting reagent.
 88. The kit of claim 87, wherein the at least one immunoglobulin detecting reagent is further defined as an anti-immunoglobulin antibody.
 89. The kit of claim 88, wherein the at least one anti-immunoglobulin antibody is preset in the first reagent at a known concentration.
 90. The kit of claim 87, further defined as comprising as a second reagent a positive sample comprising at least one immunoglobulin which binds to an allergen.
 91. The kit of claim 87, further defined as a kit for the detection of an immunoglobulin which binds to an allergen in a sample. 